Phosphate use efficiency

ABSTRACT

Isolated polynucleotides and polypeptides encoded thereby are described, together with the use of those products for making transgenic plants with increased tolerance to pH or increased phosphorus efficiency.

This application is a Continuation of co-pending application Ser. No.11/140,347, filed on May 27, 2005, the entire contents of which arehereby incorporated by reference and for which priority is claimed under35 U.S.C. §120.

Co-pending application Ser. No. 11/140,347 claims priority under 35U.S.C. §119(e) on U.S. Provisional Application No(s). 60/575,309 filedon May 27, 2004, the entire contents of which are hereby incorporated byreference.

FIELD OF THE INVENTION

The present invention relates to isolated polynucleotides, polypeptidesencoded thereby, and the use of those sequences for making transgenicplants with modulated pH response and phosphate use efficiency.

BACKGROUND OF THE INVENTION

Plants are constantly exposed to a variety of biotic (i.e., pathogeninfection and insect herbivory) and abiotic (e.g., high pH, lowphosphate) stresses. To survive these challenges, plants have developedelaborate mechanisms to perceive external signals and environmentalstresses and to manifest adaptive responses with proper physiologicaland morphological changes (Bohnert et al., 1995). Plants exposed to lowor high pH conditions typically have low yields of plant material,seeds, fruit and other edible products. Extreme soil pH conditions havea major influence on nutrient availability resulting in severe agronomiclosses. Plants exposed to low pH soil conditions develop deficiencies innutrients such as copper, molybdate, potassium, sulfur, and nitrogen.Also, plants exposed to high pH soil conditions develop iron, copper,manganese, and zinc deficiencies (FIG. 1). Phosphate deficiency is aproblem in both high and low pH soil conditions. Essential mineralnutrients are required in substantial amounts to sustain plant growthand maximize plant yields.

Consequently, agricultural and horticultural entities routinely alterthe rhizosphere to maximize and maintain crop yields; these frequentlyresult in more pollution and unbalancing of the natural soil mineralbalance (National Research Council. (1989) Alternative Agriculture.National Academic Press, Washington D.C.). Excessive over-liming of acidsoils, for instance, has resulted in the induction of iron, manganese,copper, and zinc deficiencies; deficiencies commonly observed incalcareous soil.

It would, therefore, be of great interest and importance to be able toidentify genes that confer improved phosphate efficiency characteristicsto thereby enable one to create transformed plants (such as crop plants)with improved phosphate efficiency characteristics to thereby bettersurvive low and high pH conditions.

In the field of agriculture and forestry efforts are constantly beingmade to produce plants with an increased growth potential in order tofeed the ever-increasing world population and to guarantee the supply ofreproducible raw materials. This is done conventionally through plantbreeding. The breeding process is, however, both time-consuming andlabor-intensive. Furthermore, appropriate breeding programs must beperformed for each relevant plant species.

Progress has been made in part by the genetic manipulation of plants;that is by introducing and expressing recombinant nucleic acid moleculesin plants. Such approaches have the advantage of not usually beinglimited to one plant species, but instead being transferable among plantspecies. (Zhang et al. (2004) Plant Physiol. 135:615). There is a needfor generally applicable processes that improve forest or agriculturalplant growth potential. Therefore, the present invention relates to aprocess for increasing the abiotic stress tolerance and consequently thegrowth potential in plants, characterized by expression of recombinantDNA molecules stably integrated into the plant genome.

SUMMARY OF THE INVENTION

The present invention, therefore, relates to isolated polynucleotides,polypeptides encoded thereby, and the use of those sequences for makingtransgenic plants with modulated pH tolerance or phosphate useefficiency.

The present invention also relates to processes for increasing thegrowth potential in plants under abnormal pH or phosphate conditions,recombinant nucleic acid molecules and polypeptides used for theseprocesses and their uses, as well as to plants themselves.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the relationship between soil pH and nutrient uptake.

FIG. 2 shows pH recovery as measured by volume of seeds collected from aplant containing cDNA 1248777 compared to pH treated and un-treatedcontrols.

DETAILED DESCRIPTION OF THE INVENTION 1. Definitions

The following terms are utilized throughout this application:

-   Constitutive Promoter: Promoters referred to herein as “constitutive    promoters” actively promote transcription under most, but not    necessarily all, environmental conditions and states of development    or cell differentiation. Examples of constitutive promoters include    the cauliflower mosaic virus (CaMV) 35S transcript initiation region    and the 1′ or 2′ promoter derived from T-DNA of Agrobacterium    tumefaciens, and other transcription initiation regions from various    plant genes, such as the maize ubiquitin-1 promoter, known to those    of skill.-   Domain: Domains are fingerprints or signatures that can be used to    characterize protein families and/or parts of proteins. Such    fingerprints or signatures can comprise conserved (1) primary    sequence, (2) secondary structure, and/or (3) three-dimensional    conformation. Generally, each domain has been associated with either    a family of proteins or motifs. Typically, these families and/or    motifs have been correlated with specific in-vitro and/or in-vivo    activities. A domain can be any length, including the entirety of    the sequence of a protein. Detailed descriptions of the domains,    associated families and motifs, and correlated activities of the    polypeptides of the instant invention are described below. Usually,    the polypeptides with designated domain(s) can exhibit at least one    activity that is exhibited by any polypeptide that comprises the    same domain(s).-   Endogenous: The term “endogenous,” within the context of the current    invention refers to any polynucleotide, polypeptide or protein    sequence which is a natural part of a cell or organisms regenerated    from said cell.-   Exogenous: “Exogenous,” as referred to within, is any    polynucleotide, polypeptide or protein sequence, whether chimeric or    not, that is initially or subsequently introduced into the genome of    an individual host cell or the organism regenerated from said host    cell by any means other than by a sexual cross. Examples of means by    which this can be accomplished are described below, and include    Agrobacterium-mediated transformation (of dicots—e.g. Salomon et al.    EMBO J. 3:141 (1984); Herrera-Estrella et al. EMBO J. 2:987 (1983);    of monocots, representative papers are those by Escudero et al.,    Plant J. 10:355 (1996), Ishida et al., Nature Biotechnology 14:745    (1996), May et al., Bio/Technology 13:486 (1995)), biolistic methods    (Armaleo et al., Current Genetics 17:97 1990)), electroporation, in    planta techniques, and the like. Such a plant containing the    exogenous nucleic acid is referred to here as a T₀ for the primary    transgenic plant and T₁ for the first generation. The term    “exogenous” as used herein is also intended to encompass inserting a    naturally found element into a non-naturally found location.-   Functionally Comparable Proteins: This phrase describes those    proteins that have at least one characteristic in common. Such    characteristics include sequence similarity, biochemical activity,    transcriptional pattern similarity and phenotypic activity.    Typically, the functionally comparable proteins share some sequence    similarity or at least one biochemical and within this definition,    homologs, orthologs and analogs are considered to be functionally    comparable. In addition, functionally comparable proteins generally    share at least one biochemical and/or phenotypic activity.

Functionally comparable proteins will give rise to the samecharacteristic to a similar, but not necessarily to the same degree.Typically, comparable proteins give the same characteristics where thequantitative measurement due to one of the comparables is at lest 20% ofthe other; more typically, between 30 to 40%; even more typically,between 50-60%; even more typically, 70 to 80%; even more typicallybetween 90 to 100%.

-   Heterologous sequences: “Heterologous sequences” are those that are    not operatively linked or are not contiguous to each other in    nature. For example, a promoter from corn is considered heterologous    to an Arabidopsis coding region sequence. Also, a promoter from a    gene encoding a growth factor from corn is considered heterologous    to a sequence encoding the corn receptor for the growth factor.    Regulatory element sequences, such as UTRs or 3′ end termination    sequences that do not originate in nature from the same gene as the    coding sequence originates from, are considered heterologous to said    coding sequence. Elements operatively linked in nature and    contiguous to each other are not heterologous to each other. On the    other hand, these same elements remain operatively linked but become    heterologous if other filler sequence is placed between them. Thus,    the promoter and coding sequences of a corn gene expressing an amino    acid transporter are not heterologous to each other, but the    promoter and coding sequence of a corn gene operatively linked in a    novel manner are heterologous.-   High pH: “High pH” can be defined as a non-optimal and terminal    alkaline pH value when a given plant can no longer make use of    certain essential nutrients, such as phosphate, available in the    soil. For instance, if a plant grows optimally at pH of 4.0-5.0,    high pH would be any pH greater than 5. If the optimal pH were in    the range of 6-6.5, high pH would be a pH greater than pH 6.5. As an    example, if a corn crop under optimal pH conditions would yield 134    bushels per acre and all other conditions were held constant, a high    pH tolerant variety would produce similar yields at pH 9 or above.-   Inducible Promoter: An “inducible promoter” in the context of the    current invention refers to a promoter which is regulated under    certain conditions, such as light, chemical concentration, protein    concentration, conditions in an organism, cell, or organelle, etc. A    typical example of an inducible promoter, which can be utilized with    the polynucleotides of the present invention, is PARSK1, the    promoter from the Arabidopsis gene encoding a serine-threonine    kinase enzyme, and which promoter is induced by dehydration,    abscissic acid and sodium chloride (Wang and Goodman, Plant 1 8:37    (1995)). Examples of environmental conditions that may affect    transcription by inducible promoters include anaerobic conditions,    elevated temperature, or the presence of light.-   Low Nitrogen: “Low nitrogen” can be defined as a quantity of    nitrogen, whether in the form of ammonium or nitrate, which is    insufficient to sustain normal growth and yield for a given plant.    The need for nitrogen fertilizers varies considerably among plants.    Further, the type of soil and the conditions in the soil have a    significant impact on the ability of a plant to take up nitrogen.    Supplemental nitrogen fertilizers are often added to soil or applied    directly to plants to enhance their growth or appearance. Even with    normal fertilizer applications, the amount of nitrogen available to    a plant at any given time may be too low to support optimal growth.    Hence, low nitrogen must be defined in terms of the specific plant    and environment in which the plant is being grown. For example, if    under a given set of conditions with a specific corn hybrid the    optimal nitrogen level was 160 pounds of nitrogen fertilizer per    acre and under such conditions the hybrid were able to achieve a    yield of 134 bushels per acre, a low nitrogen tolerant hybrid would    grow optimally and produce the same yield with at least10% less or    at least 20% less or at least 30% less or at least 40% less or at    least 50% less nitrogen. Further, the low nitrogen hybrid would grow    better after much of the initial nitrogen had been depleted and    would not require multiple applications of nitrogen.-   Low pH: “Low pH” can be defined as that non-optimal and terminal    acidic pH value when a given plant can no longer make use of certain    essential nutrients, such as potassium, available in the soil. If a    plant grows optimally at pH of 4.0-5.0, low pH is any pH less    than 4. If the optimal pH is in the range of 6-8, low pH would be a    pH less than 6. For example, if a corn crop under optimal pH    conditions would yield 134 bushels per acre and all other conditions    were held constant, a low pH tolerant variety would produce similar    yields at pH 5, or pH 4.-   Low Phosphate: “Low phosphate” can be defined as a quantity of    phosphate which is insufficient to sustain normal growth and yield    for a given plant. The level of phosphate required for optimal plant    growth differs among plant species and depends on the condition of    the soil and other environmental conditions. To determine a level of    phosphate that is low, comparative experiments are needed. For    example, if a corn hybrid in a particular field treated with 40    pounds of phosphate per acre would yield 134 bushels per acre and    all other conditions were held constant, a low phosphate tolerant    hybrid would produce similar yields at 35 or less pounds of    phosphate per acre or 30 or less pounds of phosphate per acre or 25    or less pounds of phosphate per acre or 20 or less pounds of    phosphate per acre.-   Masterpool: The “master pools” discussed in these experiments are a    pool of seeds from five different transgenic plants transformed with    the same exogenous gene.-   Misexpression: The term “misexpression” refers to an increase or a    decrease in the transcription of a coding region into a    complementary RNA sequence as compared to the wild-type. This term    also encompasses expression of a gene or coding region for a    different time period as compared to the wild-type and/or from a    non-natural location within the plant genome.-   Percentage of sequence identity: “Percentage of sequence identity,”    as used herein, is determined by comparing two optimally aligned    sequences over a comparison window, where the fragment of the    polynucleotide or amino acid sequence in the comparison window may    comprise additions or deletions (e.g., gaps or overhangs) as    compared to the reference sequence (which does not comprise    additions or deletions) for optimal alignment of the two sequences.    The percentage is calculated by determining the number of positions    at which the identical nucleic acid base or amino acid residue    occurs in both sequences to yield the number of matched positions,    dividing the number of matched positions by the total number of    positions in the window of comparison and multiplying the result by    100 to yield the percentage of sequence identity. Optimal alignment    of sequences for comparison may be conducted by the local homology    algorithm of Smith and Waterman Add. APL. Math. 2:482 (1981), by the    homology alignment algorithm of Needleman and Wunsch J. Mol. Biol.    48:443 (1970), by the search for similarity method of Pearson and    Lipman Proc. Natl. Acad. Sci. (USA) 85: 2444 (1988), by computerized    implementations of these algorithms (GAP, BESTFIT, BLAST, PASTA, and    TFASTA in the Wisconsin Genetics Software Package, Genetics Computer    Group (GCG), 575 Science Dr., Madison, Wis.), or by inspection.    Given that two sequences have been identified for comparison, GAP    and BESTFIT are preferably employed to determine their optimal    alignment. Typically, the default values of 5.00 for gap weight and    0.30 for gap weight length are used. The term “substantial sequence    identity” between polynucleotide or polypeptide sequences refers to    polynucleotide or polypeptide comprising a sequence that has at    least 80% sequence identity, preferably at least 85%, more    preferably at least 90% and most preferably at least 95%, even more    preferably, at least 96%, 97%, 98% or 99% sequence identity compared    to a reference sequence using the programs.

Query nucleic acid and amino acid sequences were searched againstsubject nucleic acid or amino acid sequences residing in public orproprietary databases. Such searches were done using the WashingtonUniversity Basic Local Alignment Search Tool Version 1.83 (WU-Blast2)program. The WU-Blast2 program is available on the internet fromWashington University. A WU-Blast2 service for Arabidopsis can also befound on the internet. Typically the following parameters of WU-Blast2were used: Filter options were set to “default,” Output format was setto “gapped alignments,” the Comparison Matrix was set to “BLOSUM62,”Cutoff Score (S value) was set to “default,” the Expect (E threshold)was set to “default,” the Number of best alignments to show was set to“100,” and the “Sort output” option was set to sort the output by“pvalue.”

-   Plant Promoter: A “plant promoter” is a promoter capable of    initiating transcription in plant cells and can drive or facilitate    transcription of a nucleotide sequence or fragment thereof of the    instant invention. Such promoters need not be of plant origin. For    example, promoters derived from plant viruses, such as the CaMV35S    promoter or from Agrobacterium tumefaciens such as the T-DNA    promoters, can be plant promoters. A typical example of a plant    promoter of plant origin is the maize ubiquitin-1 (ubi-1) promoter    known to those of skill.-   Specific Promoter: In the context of the current invention,    “specific promoters” refers to promoters that have a high preference    for being active in a specific tissue or cell and/or at a specific    time during development of an organism. By “high preference” is    meant at least 3-fold, preferably 5-fold, more preferably at least    10-fold still more preferably at least 20-fold, 50-fold or 100-fold    increase in transcription in the desired tissue over the    transcription in any other tissue. Typical examples of temporal    and/or tissue specific promoters of plant origin that can be used    with the polynucleotides of the present invention, are: SH-EP from    Vigna mungo and EP-C1 from Phaseolus vulgaris (Yamauchi et    al. (1996) Plant Mol Biol. 30(2):321-9.); RCc2 and RCc3, promoters    that direct root-specific gene transcription in rice (Xu et al.,    Plant Mol. Biol. 27:237 (1995) and TobRB27, a root-specific promoter    from tobacco (Yamamoto et al., Plant Cell 3:371 (1991)).-   Stringency: “Stringency” as used herein is a function of probe    length, probe composition (G+C content), and salt concentration,    organic solvent concentration, and temperature of hybridization or    wash conditions. Stringency is typically compared by the parameter    T_(m), which is the temperature at which 50% of the complementary    molecules in the hybridization are hybridized, in terms of a    temperature differential from T_(m). High stringency conditions are    those providing a condition of T_(m)−5° C. to T_(m)−10° C. Medium or    moderate stringency conditions are those providing T_(m)−20° C. to    T_(m)−29° C. Low stringency conditions are those providing a    condition of T_(m)−40° C. to T_(m)−48° C. The relationship of    hybridization conditions to T_(m) (in ° C.) is expressed in the    mathematical equation

T _(m)=81.5−16.6(log₁₀[Na⁺])+0.41(% G+C)−(600/N)   (1)

where N is the length of the probe. This equation works well for probes14 to 70 nucleotides in length that are identical to the targetsequence. The equation below for T_(m) of DNA-DNA hybrids is useful forprobes in the range of 50 to greater than 500 nucleotides, and forconditions that include an organic solvent (formamide).

T _(m)=81.5+16.6 log {[Na⁺]/(1+0.7[Na⁺])}±0.41(% G+C)−500/L 0.63(%formamide)   (2)

where L is the length of the probe in the hybrid. (P. Tijessen,“Hybridization with Nucleic Acid Probes” in Laboratory Techniques inBiochemistry and Molecular Biology, P. C. vand der Vliet, ed., c. 1993by Elsevier, Amsterdam.) The T_(m) of equation (2) is affected by thenature of the hybrid; for DNA-RNA hybrids T_(m) is 10-15° C. higher thancalculated, for RNA-RNA hybrids T_(m) is 20-25° C. higher. Because theT_(m) decreases about 1° C. for each 1% decrease in homology when a longprobe is used (Bonner et al., J. Mol. Biol. 81:123 (1973)), stringencyconditions can be adjusted to favor detection of identical genes orrelated family members.

Equation (2) is derived assuming equilibrium and therefore,hybridizations according to the present invention are most preferablyperformed under conditions of probe excess and for sufficient time toachieve equilibrium. The time required to reach equilibrium can beshortened by inclusion of a hybridization accelerator such as dextransulfate or another high volume polymer in the hybridization buffer.

Stringency can be controlled during the hybridization reaction or afterhybridization has occurred by altering the salt and temperatureconditions of the wash solutions used. The formulas shown above areequally valid when used to compute the stringency of a wash solution.Preferred wash solution stringencies lie within the ranges stated above;high stringency is 5-8° C. below T_(m), medium or moderate stringency is26-29° C. below T_(m) and low stringency is 45-48° C. below T_(m).

-   Superpool: As used in the context of the current invention, a    “superpool” refers to a mixture of seed from 100 different “master    pools”. Thus, the superpool contains an equal amount of seed from    500 different events, but only represents 100 transgenic plants with    a distinct exogenous nucleotide sequence transformed into them,    because the master pools are of 5 different events with the same    exogenous nucleotide sequence transformed into them.-   T₀: As used in the current application, the term “T₀” refers to the    whole plant, explant, or callous tissue inoculated with the    transformation medium.-   T₁: As used in the current application, the term T₁ refers to the    either the progeny of the T₀ plant, in the case of whole-plant    transformation, or the regenerated seedling in the case of explant    or callous tissue transformation.-   T₂: As used in the current application, the term T₂ refers to the    progeny of the T₁ plant. T₂ progeny are the result of    self-fertilization or cross pollination of a T₁ plant.-   T₃: As used in the current application, the term T₃ refers to second    generation progeny of the plant that is the direct result of a    transformation experiment. T₃ progeny are the result of    self-fertilization or cross pollination of a T₂ plant.-   Zero Nitrogen: Nitrogen is not present in any amount.-   Zero Phosphorus: Phosphorus is not present in any amount.

2. Important Characteristics of the Polynucleotides and Polypeptides ofthe Invention

The polynucleotides and polypeptides of the present invention are ofinterest because when they are misexpressed (i.e. when expressed at anon-natural location or in an increased or decreased amount) theyproduce plants with modified pH tolerance or phosphate use efficiency.“Phosphate use efficiency” is a term that includes various responses toenvironmental conditions that affect the amount of phosphate availableto the plant. For example, under both low and high pH conditionsphosphate is bound within the soil, resulting in a decrease of availablephosphate for maintaining or initiating physiological processes. As usedherein, modulating phosphate use efficiency is intended to encompass allof these situations as well as other environmental situations thataffect the plant's ability to use and/or maintain phosphate effectively(e.g. osmotic stress, etc.).

The polynucleotides and polypeptides of the invention, as discussedbelow and as evidenced by the results of various experiments, are usefulfor modulating pH tolerance or phosphate use efficiency. These traitscan be used to exploit or maximize plant products for agricultural,ornamental or forestry purposes in different environment conditions ofwater supply. Modulating the expression of the nucleotides andpolypeptides of the present invention leads to transgenic plants thatwill be less sensitive to variations in pH and that require lessphosphate, resulting in better yields under these types of adverseconditions. Both categories of transgenic plants lead to reduced costsfor the farmer and better yield in their respective environmentalconditions.

3. The Polynucleotides and Polypeptides of the Invention

The polynucleotides of the invention, and the proteins expressedthereby, are set forth in the sequences present in the Sequence Listing.Some of these sequences are functionally comparable proteins.

Functionally comparable proteins are those proteins that have at leastone characteristic in common. Such characteristics can include sequencesimilarity, biochemical activity and phenotypic activity. Typically, thefunctionally comparable proteins share some sequence similarity andgenerally share at least one biochemical and/or phenotypic activity. Forexample, biochemical functionally comparable proteins are proteins thatact on the same reactant to give the same product.

Another class of functionally comparable proteins is phenotypicfunctionally comparable proteins. The members of this class regulate thesame physical characteristic, such as increased drought tolerance.Proteins can be considered phenotypic functionally comparable proteinseven if the proteins give rise to the same physical characteristic, butto a different degree.

The polypeptides of the invention also include those comprising theconsensus sequences described in Tables 1-5, 2-6 and 3-5. A consensussequence defines the important conserved amino acids and/or domainswithin a polypeptide. Thus, all those sequences that conform to theconsensus sequence are suitable for the same purpose. Polypeptidescomprised of a sequence within and defined by one of the consensussequences can be utilized for the purposes of the invention namely tomake transgenic plants with improved tolerance to heat or high or lowwater conditions.

4. Use of the Polynucleotides and Polypeptides to Make Transgenic Plants

To use the sequences of the present invention or a combination of themor parts and/or mutants and/or fusions and/or variants of them,recombinant DNA constructs are prepared which comprise thepolynucleotide sequences of the invention inserted into a vector, andwhich are suitable for transformation of plant cells. The construct canbe made using standard recombinant DNA techniques (Sambrook et al. 1989)and can be introduced to the species of interest byAgrobacterium-mediated transformation or by other means oftransformation as referenced below.

The vector backbone can be any of those typical in the art such asplasmids, viruses, artificial chromosomes, BACs, YACs and PACs andvectors of the sort described by

-   (a) BAC: Shizuya et al., Proc. Natl. Acad. Sci. USA 89: 8794-8797    (1992); Hamilton et al., Proc. Natl. Acad. Sci. USA 93: 9975-9979    (1996);-   (b) YAC: Burke et al., Science 236:806-812 (1987);-   (c) PAC: Sternberg N. et al., Proc Natl Acad Sci USA. January;    87(1):103-7 (1990);-   (d) Bacteria-Yeast Shuttle Vectors: Bradshaw et al., Nucl Acids Res    23: 4850-4856 (1995);-   (e) Lambda Phage Vectors: Replacement Vector, e.g., Frischauf et    al., J. Mol Biol 170: 827-842 (1983); or Insertion vector, e.g.,    Huynh et al., In: Glover N M (ed) DNA Cloning: A practical Approach,    Vol. 1 Oxford: IRL Press (1985); T-DNA gene fusion vectors :Walden    et al., Mol Cell Biol 1: 175-194 (1990); and-   (g) Plasmid vectors: Sambrook et al., infra.

Typically, the construct comprises a vector containing a sequence of thepresent invention with any desired transcriptional and/or translationalregulatory sequences, such as promoters, UTRs, and 3′ end terminationsequences. Vectors can also include origins of replication, scaffoldattachment regions (SARs), markers, homologous sequences, introns, etc.The vector may also comprise a marker gene that confers a selectablephenotype on plant cells. The marker typically encodes biocideresistance, particularly antibiotic resistance, such as resistance tokanamycin, bleomycin, hygromycin, or herbicide resistance, such asresistance to glyphosate, chlorosulfuron or phosphinotricin.

A plant promoter is used that directs transcription of the gene in alltissues of a regenerated plant and may be a constitutive promoter, suchas p326 or CaMV35S. Alternatively, the plant promoter directstranscription of a sequence of the invention in a specific tissue manner(tissue-specific promoter) or is otherwise under more preciseenvironmental control (inducible promoter). Various plant promoters,including constitutive, tissue-specific and inducible, are known tothose skilled in the art and can be utilized in the present invention.Typically, preferred promoters to use in the present invention are thosethat are induced by heat or low water conditions Such as the RD29apromoter (Kasuga et al., Plant Cell Physiol. 45:346 (2004) andYamaguchi-Shinozaki and Shinozaki, Mol Gen Genet. 236: 331 (1993)) orother DRE-containing (dehydration-responsive elements) promoters (Liu etal, Cell 10: 1391 (1998)). Another preferred embodiment of the presentinvention is the use of root specific promoters such as those present inthe AtXTH17, AtXTH18, AtXTH19 and AtXTH20 genes of Arabidopsis(Vissenberg et al. (2005) Plant Cell Physiol 46:192) or guard cellspecific promoters such as TGG1 or KST1 (Husebye et al. (2002) PlantPhysiol 128:1180; Plesch et al. (2001) Plant J 28:455).

Alternatively, misexpression can be accomplished using a two componentsystem, whereby the first component comprises a transgenic plantcomprising a transcriptional activator operatively linked to a promoterand the second component comprises a transgenic plant comprising asequence of the invention operatively linked to the target bindingsequence/region of the transcriptional activator. The two transgenicplants are crossed and the sequence of the invention is expressed intheir progeny. In another alternative, the misexpression can beaccomplished by transforming the sequences of the two component systeminto one transgenic plant line.

Any promoter that functions in plants can be used in the firstcomponent, such as those discussed above. Suitable transcriptionalactivator polypeptides include, but are not limited to, those encodingHAP1 and GAL4. The binding sequence recognized and targeted by theselected transcriptional activator protein (e.g. a UAS element) is usedin the second component.

Transformation

Nucleotide sequences of the invention are introduced into the genome orthe cell of the appropriate host plant by a variety of techniques. Thesetechniques for transforming a wide variety of higher plant species arewell known and described in the technical and scientific literature.See, e.g. Weising et al., Ann. Rev. Genet. 22:421 (1988); and Christou,Euphytica, v. 85, n.1-3:13-27, (1995).

Processes for the transformation and regeneration of monocotyledonousand dicotyledonous plants are known to the person skilled in the art.For the introduction of DNA into a plant host cell a variety oftechniques is available. These techniques include transformation ofplant cells by injection (e.g. Newell, 2000), microinjection (e.g.Griesbach (1987) Plant Sci. 50 69-77), electroporation of DNA (e.g.Fromm et al. (1985) Proc. Natl Acad. Sci. USA 82:5824 and Wan andLemaux, Plant Physiol. 104 (1994), 37-48), PEG (e.g. Paszkowski et al.(1984) EMBO J. 3:2717), use of biolistics (e.g. Klein et al. (1987)Nature 327:773), fusion of cells or protoplasts (Willmitzer, L., 1993Transgenic plants. In: Biotechnology, A Multi-Volume ComprehensiveTreatise (H. J. Rehm, G. Reed, A. Pühler, P. Stadler, eds., Vol. 2,627-659, VCH Weinheim-New York-Basel-Cambridge), via T-DNA usingAgrobacterium tumefaciens (e.g. Fraley et al. (Crit. Rev. Plant. Sci. 4,1-46 and Fromm et al., Biotechnology 8 (1990), 833-844) or Agrobacteriumrhizogenes (e.g. Cho et al. (2000) Planta 210:195-204) or otherbacterial hosts (e.g. Brootghaerts et al. (2005) Nature 433:629-633), aswell as further possibilities.

In addition, a number of non-stable transformation methods well known tothose skilled in the art may be desirable for the present invention.Such methods include, but are not limited to, transient expression (e.g.Lincoln et al. (1998) Plant Mol. Biol. Rep. 16:1-4) and viraltransfection (e.g. Lacomme et al. (2001) In “Genetically EngineeredViruses” (C. J. A. Ring and E. D. Blair, Eds). Pp. 59-99, BIOSScientific Publishers, Ltd. Oxford, UK).

Seeds are obtained from the transformed plants and used for testingstability and inheritance. Generally, two or more generations arecultivated to ensure that the phenotypic feature is stably maintainedand transmitted.

One of skill will recognize that after the expression cassette is stablyincorporated in transgenic plants and confirmed to be operable, it canbe introduced into other plants by sexual crossing. Any of a number ofstandard breeding techniques can be used, depending upon the species tobe crossed.

The nucleic acids of the invention can be used to confer the trait ofincreased tolerance to heat and/or low water conditions, withoutreduction in fertility, on essentially any plant.

The nucleotide sequences according to the invention encode appropriateproteins from any organism, in particular from plants, fungi, bacteriaor animals.

The process according to the invention can be applied to any plant,preferably higher plants, pertaining to the classes of Angiospermae andGymnospermae. Plants of the subclasses of the Dicotylodenae and theMonocotyledonae are particularly suitable. Dicotyledonous plants belongto the orders of the Magniolales, Illiciales, Laurales, PiperalesAristochiales, Nymphaeales, Ranunculales, Papeverales, Sarraceniaceae,Trochodendrales, Hamamelidales, Eucomiales, Leitneriales, Myricales,Fagales, Casuarinales, Caryophyllales, Batales, Polygonales,Plumbaginales, Dilleniales, Theales, Malvales, Urticales, Lecythidales,Violales, Salicales, Capparales, Ericales, Diapensales, Ebenales,Primulales, Rosales, Fabales, Podostemales, Haloragales, Myrtales,Comales, Proteales, Santales, Rafflesiales, Celastrales, Euphorbiales,Rhanmales, Sapindales, Juglandales, Geraniales, Polygalales, Umbellales,Gentianales, Polemoniales, Lamiales, Plantaginales, Scrophulariales,Campanulales, Rubiales, Dipsacales, and Asterales. Monocotyledonousplants belong to the orders of the Alismatales, Hydrocharitales,Najadales, Triuridales, Commelinales, Eriocaulales, Restionales, Poales,Juncales, Cyperales, Typhales, Bromeliales, Zingiberales, Arecales,Cyclanthales, Pandanales, Arales, Lilliales, and Orchidales. Plantsbelonging to the class of the Gymnospermae are Pinales, Ginkgoales,Cycadales and Gnetales.

The method of the invention is preferably used with plants that areinteresting for agriculture, horticulture, biomass for bioconversionand/or forestry. Examples are tobacco, oilseed rape, sugar beet, potato,tomato, cucumber, pepper, bean, pea, citrus fruit, apple, pear, berries,plum, melon, eggplant, cotton, soybean, sunflower, rose, poinsettia,petunia, guayule, cabbage, spinach, alfalfa, artichoke, corn, wheat,rye, barley, grasses such as switch grass or turf grass, millet, hemp,banana, poplar, eucalyptus trees, conifers.

Homologs Encompassed by the Invention

Agents of the invention include proteins comprising at least about acontiguous 10 amino acid region preferably comprising at least about acontiguous 20 amino acid region, even more preferably comprising atleast about a contiguous 25, 35, 50, 75 or 100 amino acid region of aprotein of the present invention. In another preferred embodiment, theproteins of the present invention include between about 10 and about 25contiguous amino acid region, more preferably between about 20 and about50 contiguous amino acid region, and even more preferably between about40 and about 80 contiguous amino acid region.

Due to the degeneracy of the genetic code, different nucleotide codonsmay be used to code for a particular amino acid. A host cell oftendisplays a preferred pattern of codon usage. Nucleic acid sequences arepreferably constructed to utilize the codon usage pattern of theparticular host cell. This generally enhances the expression of thenucleic acid sequence in a transformed host cell. Any of the abovedescribed nucleic acid and amino acid sequences may be modified toreflect the preferred codon usage of a host cell or organism in whichthey are contained. Modification of a nucleic acid sequence for optimalcodon usage in plants is described in U.S. Pat. No. 5,689,052.Additional variations in the nucleic acid sequences may encode proteinshaving equivalent or superior characteristics when compared to theproteins from which they are engineered.

It is understood that certain amino acids may be substituted for otheramino acids in a protein or peptide structure (and the nucleic acidsequence that codes for it) without appreciable change or loss of itsbiological utility or activity. The amino acid changes may be achievedby changing the codons of the nucleic acid sequence.

It is well known in the art that one or more amino acids in a nativesequence can be substituted with other amino acid(s), the charge andpolarity of which are similar to that of the native amino acid, i.e., aconservative amino acid substitution, resulting in a silent change.Conservative substitutes for an amino acid within the native polypeptidesequence can be selected from other members of the class to which theamino acid belongs (see below). Amino acids can be divided into thefollowing four groups: (1) acidic (negatively charged) amino acids, suchas aspartic acid and glutamic acid; (2) basic (positively charged) aminoacids, such as arginine, histidine, and lysine; (3) neutral polar aminoacids, such as glycine, serine, threonine, cysteine, cystine, tyrosine,asparagine, and glutamine; and (4) neutral nonpolar (hydrophobic) aminoacids such as alanine, leucine, isoleucine, valine, proline,phenylalanine, tryptophan, and methionine.

In a further aspect of the present invention, nucleic acid molecules ofthe present invention can comprise sequences that differ from thoseencoding a protein or fragment thereof selected from the groupconsisting of those sequences present in the Sequence Listing due to thefact that the different nucleic acid sequence encodes a protein havingone or more conservative amino acid changes.

In another aspect, biologically functional equivalents of the proteinsor fragments thereof of the present invention can have about 10 or fewerconservative amino acid changes, more preferably about 7 or fewerconservative amino acid changes, and most preferably about 5 or fewerconservative amino acid changes. In a preferred embodiment, the proteinhas between about 5 and about 500 conservative changes, more preferablybetween about 10 and about 300 conservative changes, even morepreferably between about 25 and about 150 conservative changes, and mostpreferably between about 5 and about 25 conservative changes or between1 and about 5 conservative changes.

5. Experiments Confirming the Usefulness of the Polynucleotides andPolypeptides of the Invention

5.1 Procedures

The nucleotide sequences of the invention were identified by use of avariety of screens for pH and/or low phosphate and/or low nitrogenconditions. These screens are recognized by those skilled in the art tobe predictive of nucleotide sequences that provide plants with improvedtolerance to pH and/or low phosphate and/or low nitrogen conditionsbecause they emulate the different environmental conditions that canresult from increased pH and/or low phosphate and/or low nitrogenconditions. These screens generally fall into two categories (1) soilscreens and (2) in vitro screens.

Soil screens have the advantage of assaying the response of the entireplant to particular conditions, such as high pH or low phosphorus. Onthe other hand, in vitro screens have the advantage of relying ondefined media and so allow more defined manipulation of growthconditions. Each of the screens used is described in more detail below.

In general, the screens used to identify the polynucleotides andpolypeptides of the invention were conducted using superpools ofArabidopsis T₂ transformed plants. The T₁ plants were transformed with aTi plasmid containing a particular SEQ ID NO in the sense orientationrelative to a constitutive promoter and harboring the plant-selectablemarker gene phosphinothricin acetyltansferase (PAT), which confersherbicide resistance to transformed plants. For in vitro screens, seedfrom multiple superpools (1,200 T₂ seeds from each superpool) wereusually tested. T₃ seed were collected from the resistant plants andretested on one or more in vitro screens. The results of the screensconducted for each SEQ ID NO can be found in the Examples below.

1. High pH

Screens for high pH resistance identify seedlings better able to thriveunder nutritional deficiencies (e.g. Phosphate, Manganese, Iron, Boron)imposed by alkaline conditions.

Seeds are sterilized in 50% household bleach for 5 minutes and thenwashed with double distilled deionized water three times. Sterilizedseed is stored in the dark at 4° C. for a minimum of 3 days before use.

High pH media is prepared by mixing 0.5 g/l MES hydrate with 1X MS+0.5%Sucrose. Prior to autoclaving pH is adjusted with 10 N KNH to thefollowing values: pH 5.7 (control), pH 7.03, pH 8.02, pH 9.01 and pH10.18. The media pH is retested since pH values drop after autoclavingas follows: pH 5.7→pH 5.66; pH 7.03→pH6.50; pH 8.02→pH 7.50; pH 9.01→pH8.91; pH10.18→pH 9.91. Generally speaking, pH 9.01(pH 8.91) allowsgermination but no growth beyond 2 to 5 mm and no root growth.Germination does not occur at higher pH (e.g. pH 10.81).

Approximately 1200 seeds are evenly spaced per MS-sucrose plate beforeincubating in the vertical position at 22° C. for 14 days. Under theseconditions, the plates are exposed to 12,030 LUX from above and 3,190LUX from the bottom.

Seedlings are scored for root and shoot growth after 7 and 14 days.Putative tolerant seedlings are transferred to MS pH 5.7 for recoveryfor 14 days prior to transplanting in soil. Finale™ spraying is doneafter plants are moved to soil to remove non-transgenics from thepopulation.

DNA is isolated from each T₂ plant and used in PCR reactions using thefollowing cycling conditions: 95° C. for 5 min, 35 cycles of (94° C. for30 sec, then 59° C. for 30 sec, then 72° C. for 1 min), 72° C. for 8 minand 4° C. hold. Aliquots of the reaction product are analyzed on a 1.0%agarose gel stained with ethidium bromide. The DNA products aresequenced to determine which insert sequences were in each superpoolcandidate chosen in the screen.

T₃ Seed from those plants containing sequenced PCR products arecollected and retested on high pH media. In addition, plants are testedon MS media lacking Phosphate and having a pH of 5.7.

2. Zero Phosphate

Screens for zero phosphate tolerance identify seedlings better able tothrive under a phosphate nutritional deficiency.

Seeds are sterilized in 50% household bleach for 5 minutes and thenwashed with double distilled deionized water three times. Sterilizedseed is stored in the dark at 4° C. for a miniumum of 3 days before use.

Zero phosphate media is prepared using commercially available MS medialacking phosphate, pH 5.7.

Approximately 1200 seeds are evenly spaced per MS-P plate beforeincubating in the vertical position at 22° C. for 14 days. Under theseconditions, the plates are exposed to 12,030 LUX from above and 3,190LUX from the bottom.

Seedlings are scored for root and shoot growth after 7 and 14 days.Putative tolerant seedlings are transferred to MS pH 5.7 for recoveryfor 14 days prior to transplanting in soil. Finale™ spraying is doneafter the plants are moved to soil to remove non-transgenics from thepopulation.

DNA is isolated from each T₂ plant and used in PCR reactions using thefollowing cycling conditions: 95° C. for 5 min, 35 cycles of (94° C. for30 sec, then 59° C. for 30 sec, then 72° C. for 1 min), 72° C. for 8 minand 4° C. hold. Aliquots of the reaction product are analyzed on a 1.0%agarose gel stained with ethidium bromide. The DNA products aresequenced to determined which insert sequences were in each superpoolcandidate chosen in the screen.

T₃ Seed from those plants containing the sequenced PCR products arecollected and retested.

3. Zero Phosphate, Zero Nitrogen

Screens for zero phosphate, zero nitrogen tolerance identify seedlingsbetter able to thrive under a phosphate nutritional deficiency.

Seeds are sterilized in 50% household bleach for 5 minutes and thenwashed with double distilled deionized water three times. Sterilizedseed is stored in the dark at 4° C. for a miniumum of 3 days before use.

Zero phosphate, zero nitrogen media is prepared using commerciallyavailable MS media lacking phosphate, pH 5.7.

Approximately 1200 seeds are evenly spaced per MS-P-N plate beforeincubating in the vertical position at 22° C. for 14 days. Under theseconditions, the plates are exposed to 12,030 LUX from above and 3,190LUX from the bottom.

Growth and overall greenness are assayed 10 days post-treatment.Seedling recovery is assessed by adding a thin layer (8.3 ml) ofcomplete MS+P+N media, pH 5.7, softened by the addition of 0.02% agar.Media is added to the edge of the plate and slowly rotated until a thinfilm of +PN media is present on top of the solidified −PN media.Putative tolerant seedlings are greener and have increased growthcompared to controls. Finale™ spraying is done after the plants aremoved to soil to remove non-transgenics from the population.

DNA is isolated from each T₂ plant and used in PCR reactions using thefollowing cycling conditions: 95° C. for 5 min, 35 cycles of (94° C. for30 sec, then 59° C. for 30 sec, then 72° C. for 1 min), 72° C. for 8 minand 4° C. hold. Aliquots of the reaction product are analyzed on a 1.0%agarose gel stained with ethidium bromide. The DNA products aresequenced to determined which insert sequences were in each superpoolcandidate chosen in the screen.

T₃ Seed from those plants containing the sequenced PCR products arecollected and retested.

5.2 Results

The results of the above experiments are set forth below wherein eachindividual example relates to all of the experimental results for aparticular polynucleotide/polypeptide if the invention.

Example 1 Ceres cDNA 12335629

Clone 40781, Ceres cDNA 12335629, encodes a full-length protein withhomology to a ferredoxin thioredoxin reductase from Arabidopsisthaliana.

Ectopic expression of Ceres cDNA 12335629 under the control of theCaMV35S promoter induces the following phenotypes:

-   -   Better growth and recovery after exposure to high pH conditions        and    -   Continued growth under high pH induced phosphate and iron        deficiencies.        Generation and Phenotypic Evaluation of T₁ Lines Containing        35S::cDNA 12335629.

Wild-type Arabidopsis Wassilewskija (WS) plants were transformed with aTi plasmid containing cDNA 12335629 in the sense orientation relative tothe 35S constitutive promoter. The T_(i) plasmid vector used for thisconstruct, CRS338, contains PAT and confers herbicide resistance totransformed plants. Ten independently transformed events were selectedand evaluated for their qualitative phenotype in the T_(i) generation.No positive or negative phenotypes were observed in the T₁ plants.

Screens of Superpools on High pH Media for pH Tolerance.

Seed from superpools of the 35S over-expression lines were evaluated forgreenness and size on high pH media as described above. Once cDNA12335629 was identified in tolerant plants, the five individual T₂events containing this cDNA (ME03527) were screened on high pH mediaessentially as described above, but where the media pH is 8.5, toidentify events with the tolerant phenotype.

Results:

Qualitative Analysis of the Superpool Containing 35S::clone 40781 Plantson high pH

The screen resulted in a decrease in germination and/or growth for bothwildtype and superpools as compared to seeds on control media. Only oneline survived transplantation to soil. The candidate was greener thancontrols but overall size was comparable to those of wild-type. Therewas no delay in flowering time or decrease in seed set in comparison toun-treated wild-type but a faster flowering time and greater seed setwas apparent when compared to a recovered pH treated wild-type plant(data not shown). These results are consistent with those of the T₁generation which displayed normal flowering time and fertility.

Qualitative and Quantitative Analysis of T₃-cDNA 12335629 on High pH.

The plants were treated with Finale™ to eliminate any false-positives orany lines where the Finale™ marker was suppressed. All of theFinale™-resistant candidates flowered and set seed. Finale™ segregationwas assessed to identify events containing a single insert segregationin a 3:1 (R:S) ratio as calculated by chi-square test. All of the eventssegregated for a single functional insert (Table 1-1). The transgenicplants were greener and slightly larger than the control under high pHstress.

TABLE 1-1 Observed and expected frequencies assuming a 3:1 ratio forhigh pH tolerance of cDNA 12335629 progeny under high pH (pH 8.5). α of0.05 Probability Event Generation Observed Expected χ² of Chi-Test pHResistant T₃ 22 29 0.926 pH Sensitive T₃ 14 7 2.778 0.054 N = 36 36 363.704Qualitative and Quantitative Analysis of cDNA 12335629 Progeny on MediaLacking Phosphate

Before testing independent T₂ events, plants containing cDNA 12335629were re-assayed for phosphate starvation tolerance by growth on mediacontaining no phosphate as described above. After seven days onlyslightly more tolerance compared to controls is observed, but cDNA12335629 seedlings are a bit larger and slightly greener than those ofthe control. Because the slight increase in size was particularlydifficult to assess, anything lower or equal to the wild-type average of0.42 cm was assessed to be sensitive and anything higher was assessed astolerant. Twenty-four resistant and twelve phosphate starved sensitiveseedlings were compared to Finale™]frequencies and found to have aChi-test probability of 0.49, suggesting a positive fit (Table 1-2).

TABLE 1-2 Observed and expected frequencies assuming a 3:1 ratio forphosphate starvation tolerance among progeny of cDNA 12335629 medialacking phosphate (-P). α of 0.05 Probability Event Generation ObservedExpected χ² of Chi-Test -P Resistant T₃ 24 27 0.333 -P Sensitive T₃ 12 91.333 0.25 N = 36 36 36 1.666Qualitative and Quantitative Analysis of Individual T₂ Events of cDNA12335629 on High pH Plate Assay.

Five individual events of cDNA 12335629 (ME03527) were analyzed for apositive phenotype under high pH conditions. All five T₂ events hadwild-type germination frequencies on MS pH 5.7 plates (data not shown).All T₂ lines and recovered T₃ lines showed evidence of a single insertas determined by Chi-square analysis (Table 1-3). Seeds from each of thefive independent T₂ events, were plated on pH 8.5 plates and allowed togerminate and grow for 14 days.

Four of five T₂ events of ME03527 (-02,-03,-04, and -05) had a positivehigh pH tolerance phenotype as defined by growth and greenness. Thephenotype of1V1E03527-01 was too weak to assess as positive compared tothe controls (Table 1-4). Phenotype strength varied among the fourpositive independent events, but all showed better growth than controls.The segregation ratios, determined by a Chi-square test, show that thesegregation of the transgene is the same as observed for Finale™ (Table1-4). ME03527-02,-03,-04, and -05 had the strongest and most consistentpH tolerance phenotypes.

TABLE 1-3 Observed and expected frequencies assuming a 3:1 (R:S) ratiofor Finale ™ resistance among 35S::clone 40781 T₂ and T₃ events testedfor growth under high pH conditions. α of 0.05 Event Generation ObservedExpected χ² Probability of Chi-Test ME03527-01 Finale ™ T₂ 16 18 0.222Resistant ME03527-01 Finale ™ T₂ 8 6 0.667 0.35 Sensitive N = 24 24 240.889 ME03527-02 Finale ™ T₂ 28 27 0.037 Resistant ME03527-02 Finale ™T₂ 8 9 0.111 0.70 Sensitive N = 36 36 36 0.148 ME03527-03 Finale ™ T₂ 1718 0.056 Resistant ME03527-03 Finale ™ T₂ 7 6 0.167 0.64 Sensitive N =24 24 24 0.223 ME03527-04 Finale ™ T₂ 27 27 0 Resistant ME03527-04Finale ™ T₂ 9 9 0 1.0 Sensitive N = 36 36 36 0 ME03527-05 Finale ™ T₂ 2327 0.593 Resistant ME03527-05 Finale ™ T₂ 13 9 1.778 0.12 Sensitive N =36 36 36 2.371 cDNA 12335629 Finale ™ T₃ 22 27 0.926 Resistant cDNA12335629 Finale ™ T₃ 14 9 2.778 0.054 Sensitive N = 36 36 36 3.704

TABLE 1-4 Observed and expected frequencies of high pH toleranceassuming segregation of transgene is the same as observed in Finale ™resistance among 35S::clone 40781 T₂ and T₃ events that showed increasedgrowth under high pH conditions. α of 0.05 Event Generation ObservedExpected χ² Probability of Chi-Test ME03527-01 pH Resistant T₂ 15 25.54.324 ME03527-01 pH Sensitive T₂ 19 85.5 2.970 32E−05 N = 36 34 34 7.294ME03527-02 pH Resistant T₂ 23 24.75 0.124 ME03527-02 pH Sensitive T₂ 108.25 0.371 0.48 N = 36 33 33 0.495 ME03527-03 pH Resistant T₂ 23 23.250.003 0.92 ME03527-03 pH Sensitive T₂ 8 7.75 0.008 N = 36 31 31 0.011ME03527-04 pH Resistant T₂ 24 27 0.333 0.25 ME03527-04 pH Sensitive T₂12 9 1.000 N = 36 36 36 1.333 ME03527-05 pH Resistant T₂ 19 27 2.3700.002 ME03527-05 pH Sensitive T₂ 17 9 7.111 N = 36 36 3 9.481 cDNA12335629 pH T₃ 19 27 2.370 0.002 Resistant cDNA 12335629 pH T₃ 17 97.111 Sensitive N = 36 36 36 9.481Table 1-5 provides the results of the consensus sequence analysis basedon Ceres cDNA 13487605.

TABLE 1-5

Example 2 Ceres cDNA 12330185

Clone 34035, Ceres cDNA 12330185, encodes a 128 amino acid protein ofunknown function (DUF423) from Arabidopsis thaliana.

Ectopic expression of Ceres cDNA 12330185 under the control of the 32449promoter induces the following phenotypes:

-   -   Increased size and greenness on nutrient deficiencies incurred        by high pH conditions,    -   Better soil recovery after exposure to high pH stress, and    -   Better recovery after exposure to conditions lacking both        phosphate and nitrogen.        Generation and Phenotypic Evaluation of T₁ Lines Containing        p32449::cDNA 12330185.

Wild-type Arabidopsis Wassilewskija (WS) plants were transformed with aTi plasmid containing cDNA 12330185 in the sense orientation relative tothe 32449 constitutive promoter. Promoter 32449 has broad expressionthroughout Arabidopsis, although at much lower expression level thanCaMV35S. The T_(i) plasmid vector used for this construct, CRS311,contains PAT and confers herbicide resistance to transformed plants.Nine independently transformed events were selected and evaluated fortheir qualitative phenotype in the T₁ generation. No positive ornegative phenotypes were observed in the T₁ plants.

Screens of Superpools on High pH Media for pH Tolerance.

Seed from superpools of the 32449 over-expression lines were evaluatedfor greenness and size on high pH media as described above. Once cDNA12330185 was identified in tolerant plants, nine individual T₂ eventscontaining this cDNA (ME00077) were screened on high pH mediaessentially as described above, but where the media pH is 8.5, toidentify events with the tolerant phenotype.

Results:

Qualitative Analysis of the Superpool Containing 34449::cDNA 12330185 onHigh pH

The cDNA 12330185 line displayed a delayed flowering time of ˜8 days anddecreased seed set in comparison to the un-treated wild-type. HowevercDNA 12330185 displayed a faster flowering time (˜15 days) and greaterseed set when compared to the high pH grown wild-type plant.

Qualitative and Quantitative Analysis of the T₃ 32449:: cDNA 12330185 onHigh pH.

The cDNA 12330185 line was tested for Finale™ resistance and re-assayedfor continued pH tolerance. The segregation ratio of T₃ seeds from cDNA12330185 is suggestive of a single insert, as calculated by a Chi-squaretest (Table 2-1). The cDNA 12330185 line was re-tested on pH 9.0 mediaas described and found to be tolerant to high pH when compared tocontrols.

TABLE 2-1 Chi-square analysis of progeny of cDNA 12330185 on Finale ™assuming a 3:1 ratio. Event Observed Expected χ² Probability of Chi-TestFinale ™ Resistant 27 27 0 Finale ™ Sensitive 9 9 0 1 N = 36 36 36 0Qualitative and Quantitative Analysis of Phosphate and NitrateStarvation of T₃ (cDNA 12330185) Plants.

To ascertain whether the pH tolerant phenotype is related to bettersurvival under nutrient starvation, T₃ seeds were assayed on MS medialacking both phosphate (—P) and nitrate (—N) (pH 5.7) as describedabove. The cDNA 12330185 line was greener and of equal size compared towild-type controls. Ten days after the addition of +NP media film, cDNA12330185 seedlings recovered more quickly than wild type. Twenty-five of36 seedlings of SP9pH1 had greater growth when compared to wild type.This increased growth frequency is suggestive of a single insert asdetermined by Chi-square analysis (Table 2-2).

TABLE 2-2 Observed and expected frequencies of no phosphate/nitrategrowth assuming segregation of transgene is 3:1 (R:S) of T₃ plants ofcDNA 12330185 that showed increased growth under high pH conditions. αof 0.05 Event Observed Expected χ² Probability of Chi-Test NP Resistant25 27 0.148 0.441 NP Sensitive 11 9 0.444 N = 36 36 36 0.592Qualitative and Quantitative Analysis of Individual T₂ Events of cDNA12330185 on High pH.

Seeds from T₂ lines representing nine individual events and containingcDNA 12330185 (ME0077-01, 02, 03, 04, 05, 06, 07, 08, 09) were plated onpH media, pH 8.5 as described above. Plates were evaluated at 7 and 12days post-plating (Table 2-3). All nine T₂ events had wild-typegermination frequencies except for ME00077-04 (Table 2-4). Thisgermination problem however was not observed when seedlings were platedonto high pH plates.

Six of the nine events showed tolerance to high pH as defined by growthand greenness. The strongest tolerance phenotypes were in ME00077-03 andME00077-05. ME00077-03 and ME00077-05 both had single inserts asdetermined by Chi-square analysis (Table 2-3).

The pH tolerant phenotype was strongest in the cDNA 12330185 T₃ linerecovered from the superpool screen. We did not do a genetic mapping ofthis line's insert to determine which event it represented. This line'sphenotype was so strong that it allowed adjacent wild-type quadrantswithin same plate to grow normally after 14-days. This is most likelydue to acidification of surrounding media by the pH tolerant line.ME00077-03,-05 T₂ plants also showed increased recovery during phosphateand nitrogen starvation assays (data not shown). However, the cDNA12330185 T₃ line recovered from the superpool phenotype was strongerthan that observed for lines ME00077-03 and -05 under −NP starvationrecovery (as noted above).

TABLE 2-3

TABLE 2-4

**Germination reduction in comparison to wild-type control and otherME00077 lines

TABLE 2-5 Observed and expected frequencies of high pH toleranceassuming segregation of transgene is the same as observed in Finale ™segregation among progeny of 32449:: cDNA 12330185 T₂ events that showedincreased growth under high pH conditions. α of 0.05 Probability EventObserved Expected χ² of Chi-Test ME00077-03 pH Resistant 26 25.5 0.0090.84 ME00077-03 pH Sensitive 8 8.5 0.029 N = 36 34 34 0.038 ME00077-05pH Resistant 29 26.25 0.288 0.28 ME00077-05 pH Sensitive 6 8.75 0.864 N= 36 35 35 1.152 cDNA 12330185 pH 31 27 0.592 0.124 Resistant cDNA12330185 pH 5 9 1.778 Sensitive N = 36 36 36 2.370Table 2-6 provides the results of the consensus sequence analysis basedon Ceres cDNA 12330185.

TABLE 2-6

Example 3 Ceres cDNA 12482777

Clone 126592, Ceres cDNA 12482777, encodes a full-length protein thathas homology to an iron/manganese superoxide dismutase from Arabidopsisthaliana.

Ectopic expression of Ceres cDNA 12482777 under the control of theCaMV35S promoter induces the following phenotypes:

-   -   Increased growth under high pH induced stress    -   Better recovery after exposure to pH stress    -   Reduced height without a reduction in harvest index.        Generation and Phenotypic Evaluation of T₁ Lines Containing        35S::cDNA 12482777.

Wild-type Arabidopsis Wassilewskija (WS) plants were transformed with aTi plasmid containing cDNA 12482777 in the sense orientation relative tothe 35S constitutive promoter. The T_(i) plasmid vector used for thisconstruct, CRS338, contains PAT and confers herbicide resistance totransformed plants. Seven independently transformed events were selectedand evaluated for their qualitative phenotype in the T₁ generation. Nonegative phenotypes were observed in the T₁ plants, although an increasein the number of branches was observed one of the events.

Screens of Superpools on High pH Media for pH Tolerance.

Seed from superpools of the 35S over-expression lines were evaluated forgreenness and size on high pH media as described above. T₃ seed werealso assayed for total seed yield, total tissue dry weight and harvestindex as described above.

Results:

Qualitative Analysis of the Superpool Containing 35S:: cDNA 12482777Plants on High pH

The screen identified a single event that was greener and the overallsize was comparable to the controls. There was no delay in floweringtime or decrease in seed set compared to un-treated wild-type. Afterrecovery, the plant containing cDNA 12482777 had significantly betterseed yield, as determined by seed volume, than controls (FIG. 2).

Qualitative and Quantitative Analysis of T₃-cDNA 12482777 on High pH.

The plants were treated with Finale™ to eliminate any false-positives orany lines where the Finale™ marker was suppressed. All of theFinale™-resistant candidates flowered and set seed. Finale™ resistancesegregation in the T₃ line suggested a segregation ratio of 1:1 (R:S) ascalculated by chi-square test (Table 3-1).

The plants were greener than the pre-pH treated control. There was notolerant effect found under low phosphate conditions (data not shown),suggesting that the tolerant response is not to the nutrientdeficiencies imposed by the high pH but rather to oxidative stressinduced by alkalinity.

TABLE 3-1 Observed and expected frequencies assuming ratio for high pHtolerance among cDNA 12335629 tested for growth under high pH (pH 9.0)assuming a 3:1 (R:S) segregation ratio. α of 0.05 Probability EventGeneration Observed Expected χ² of Chi-Test cDNA T₃ 23 27 0.593 12482777pH Resistant cDNA T₃ 13 9 1.778 0.12 12482777 pH Sensitive N = 36 36 362.371Qualitative and Quantitative Analysis of Harvest Index, Seed Yield, andPlant Height of T₃ Progeny of 35S:: cDNA 12482777.

A segregating population of 17 plants containing cDNA 12482777 wasanalyzed for harvest index and seed yield compared to wild-typepopulations. Based upon stem height measurements, the transgenicpopulation of 35S:: cDNA 12482777 (10 plants) was significantly smallerthan both internal (6 plants) and external wild-type/controlpopulations. Internal wild-types/controls were those plants segregatingfrom the T₃ population of the 35S::cDNA 12482777 line which did notcontain the insert (segregating non-transgenics). External wild-typeswere non-transgenic plants from an outside source which shared nolineage with the line being tested. External wild-types are added to theexperiment as a process control to ensure the quality of the growthconditions. Average height for transgenic plants of cDNA 12482777 was33.44 cm±0.78 versus 44.65 cm±0.70 for the internal wild-type controls.Despite this decrease in plant height, harvest index, as measured byseed weight/total plant weight remained unaffected, i.e., thesetransgenic plants still produced the same ratio of total seedweight:total plant weight (biomass) as non-transgenic controls. Thisresult means that although the total seed yield is decreased in cDNA12482777 lines, it still has the same seed proportionally as controls.The cDNA 12482777 plants had a harvest index of 56.96±2.99 compared tothe wild-type population's harvest index of 44.92±2.67 (Table 3-2A).This increase in harvest index was significant at a P-value of 0.009(Table 3-3A).

It is important to note that seed weight of cDNA 12482777 plants with alarger harvest index was 0.30977g±0.025 while the wild-type populationhad an average seed weight of 0.37155g±0.027 (Table 3-3B). cDNA 12482777has a slightly smaller seed weight than the wild-type population but notstatistically different at a P-value of 0.12 (Table 3-3B), suggestingthat the harvest index of 35S:: cDNA 12482777 is comparable to, if notgreater than, wild-type plants. This increase in harvest index is notdue to an increase in number of branches (data not shown) as observed inthe T₁ generation. Instead, the internode length between siliques isreduced compared to the internal wild-type control, suggesting that cDNA12482777 plants have more siliques per stem length.

TABLE 3-2A Descriptive statistical comparison of Harvest Index betweensegregating T₄ populations containing cDNA 12482777. Internal Wild-Harvest Index: cDNA Transgenic Harvest Index: of type 12482777 smallstature Population cDNA 12482777 Wild-type stature Population Mean56.9582619 Mean 44.91972222 Standard Error 2.990040579 Standard Error2.667294901 Median 56.68809524 Median 45.56319444 Standard Deviation9.455338527 Standard Deviation 6.533511501 Sample Variance 89.40342666Sample Variance 42.68677253 Minimum 43.41666667 Minimum 33.9375 Maximum70.11666667 Maximum 54.36666667 Sum 569.582619 Sum 269.5183333 Count 10Count 6 Confidence Level 6.763946869 Confidence Level 6.856488619(95.0%) (95.0%)

TABLE 3-2B Descriptive statistical comparison of total seed weight (g)at time of harvest between segregating T₄ populations containing cDNA12482777. Total Seed Weight (g) Total Seed Weight (g) Internal Wild- of:cDNA 12482777: Transgenic of: cDNA 12482777: type Small StaturePopulation Wild-type Stature Population Mean 0.30977 Mean 0.37155Standard Error 0.024799382 Standard Error 0.027304014 Median 0.3017Median 0.3796 Standard Deviation 0.078422531 Standard Deviation0.066880902 Sample Variance 0.006150093 Sample Variance 0.004473055Minimum 0.1956 Minimum 0.2715 Maximum 0.4207 Maximum 0.4621 Sum 3.0977Sum 2.2293 Count 10 Count 6 Confidence Level 0.056100142 ConfidenceLevel 0.070187087 (95.0%) (95.0%)

TABLE 3-4A Statistical comparison of harvest index between transgenicpopulations of clone 126592 and internal wild-type populations using at-test on two samples assuming unequal variances. cDNA 1248277 Wtstature (internal wild-type population) and cDNA 12482777 small stature(transgenic population). Harvest Index: Harvest Index cDNA 12482777 cDNA12482777 Wt stature small stature Mean 44.91972222 56.9582619 Variance42.68677253 89.40342666 Observations 6 10 Hypothesized Mean Difference 0df 14 t Stat -3.004493678 P (T <= t) one-tail 0.004733406 t Criticalone-tail 1.76130925 P (T <= t) two-tail 0.009466812 t Critical two-tail2.144788596

TABLE 3-44B Statistical comparison of seed weight between transgenicpopulation of clone 126592 and internal wild-type populations using at-test on two samples assuming unequal variances. cDNA 12482777 Wtstature (internal wild-type population) and cDNA 12482777 small stature(transgenic population) Seed Weight 12482777: Seed Weight 12482777: WTstature Small Stature Mean 0.37155 0.30977 Variance 0.0044730550.006150093 Observations 6 10 Hypothesized Mean 0 Difference df 12 tStat 1.674926201 P(T <= t) one-tail 0.059894848 t Critical one-tail1.782286745 P(T <= t) two-tail 0.119789696 t Critical two-tail2.178812792Table 3-5 provides the results of the consensus sequence analysis basedon Ceres cDNA 12482777.

TABLE 3-5

Example 4 Ceres cDNA 12333678

Clone 26006, Ceres cDNA 12333678, encodes a full-length glycosylhydrolase. Ectopic expression of Ceres cDNA 12333678 under the controlof the CaMV35S promoter induces the following phenotypes:

-   -   Germination on high concentrations of polyethylene glycol (PEG),        mannitol and abscissic acid (ABA).    -   Continued growth on high PEG, mannitol and ABA.        Generation and Phenotypic Evaluation of T₁ Lines Containing        35S::cDNA 12333678.

Wild-type Arabidopsis Wassilewskija (WS) plants were transformed with aTi plasmid containing cDNA 12333678 in the sense orientation relative tothe CaMV35S constitutive promoter. The T, plasmid vector used for thisconstruct, CRS338, contains PAT and confers herbicide resistance totransformed plants. Ten independently transformed events were selectedand evaluated for their qualitative phenotype in the T₁ generation. Nopositive or negative phenotypes were observed in the T₁ plants.

Screens of Superpools on High PEG, Mannitol and ABA as Surrogate Screensfor Drought Tolerance.

Seeds from 13 superpools (1,200 T₂ seeds from each superpool) from theCaMV35S or 32449 over-expression lines were tested on high pH media asdescribed above. T₃ seeds were collected from the tolerant plants andanalyzed for tolerance on all additional high pH screens.

Once cDNA 12333678 was identified in tolerant plants, the individual T₂events containing this cDNA (ME01334) were screened on high PEG,mannitol and ABA to identify events with the resistance phenotype.

Superpools (SP) are referred to as SP1, SP2 and so on. The letterfollowing the hyphen refers to the screen (P=PEG, M=mannitol, and A=ABA)and the number following the letter refers to a number assigned to eachplant obtained from that screen on that superpool. For example, SP1-M18is the 18^(th) plant isolated from a mannitol screen of Superpool 1.

Results:

Qualitative Assessment of MEO1334 on high pH.

Superpool 1 was screened on high pH media as described above. PCRanalyses identified ME01334 as one of the ME lines showing high pHresistance. Testing of the second generation confirmed the inheritanceof the pH resistance (data not shown).

ME01334 plants that recovered after high pH produced an exceptionallylarge number of seeds compared to wild-type controls. Additional testingconfirmed that these plants statistically produce 30-80% more seeds thaneither wild-type or transgenic control plants that are recovered fromthis screen or transferred from regular MS media.

Table 4-1 provides the results of the consensus sequence analysis basedon Ceres cDNA 12333678.

TABLE 4-1

The invention being thus described, it will be apparent to one ofordinary skill in the art that various modifications of the materialsand methods for practicing the invention can be made. Such modificationsare to be considered within the scope of the invention as defined by thefollowing claims.

Each of the references from the patent and periodical literature citedherein is hereby expressly incorporated in its entirety by suchcitation.

1. An isolated nucleic acid molecule comprising: a) a nucleic acidhaving a nucleotide sequence which encodes an amino acid sequenceexhibiting at least 85% sequence identity to any one of those sequencespresent in the Sequence Listing; b) a nucleic acid which is a complementof a nucleotide sequence according to paragraph (a); (c) a nucleic acidwhich is the reverse of the nucleotide sequence according tosubparagraph (a), such that the reverse nucleotide sequence has asequence order which is the reverse of the sequence order of thenucleotide sequence according to subparagraph (a); or (d) a nucleic acidcapable of hybridizing to a nucleic acid according to any one ofparagraphs (a)-(c), under conditions that permit formation of a nucleicacid duplex at a temperature from about 40° C. and 48° C. below themelting temperature of the nucleic acid duplex.
 2. The isolated nucleicacid molecule according to claim 1, which has the nucleotide sequenceaccording to any one of those sequences present in the Sequence Listing.3. The isolated nucleic acid molecule according to claim 1, wherein saidamino acid sequence comprises a polypeptide according to any one of theconsensus sequences set forth in Tables 1-5, 2-6, 3-5 or 4-1.
 4. Theisolated nucleic acid molecule according to claim 1, wherein said aminoacid sequence has a sequence according to any one of those sequencespresent in the Sequence Listing.
 5. A vector construct comprising: a) afirst nucleic acid having a regulatory sequence capable of causingtranscription and/or translation in a plant; and b) a second nucleicacid having the sequence of the isolated nucleic acid molecule accordingto claim 1; wherein said first and second nucleic acids are operablylinked and wherein said second nucleic acid is heterologous to anyelement in said vector construct.
 6. The vector construct according toclaim 5, wherein said first nucleic acid is native to said secondnucleic acid.
 7. The vector construct according to claim 5, wherein saidfirst nucleic acid is heterologous to said second nucleic acid.
 8. Ahost cell comprising an isolated nucleic acid molecule according toclaim 1 wherein said nucleic acid molecule is flanked by exogenoussequence.
 9. A host cell comprising a vector construct according toclaim
 5. 10. An isolated polypeptide comprising an amino acid sequenceexhibiting at least 85% sequence identity to any of those sequencespresent in the Sequence Listing.
 11. A method of introducing an isolatednucleic acid into a host cell comprising: a) providing an isolatednucleic acid molecule according to claim 1; and b) contacting saidisolated nucleic acid with said host cell under conditions that permitinsertion of said nucleic acid into said host cell.
 12. A method oftransforming a host cell that comprises contacting a host cell with avector construct according to claim
 5. 13. A method for detecting anucleic acid in a sample which comprises: a) providing an isolatednucleic acid molecule according to claim 1; b) contacting said isolatednucleic acid molecule with a sample under conditions which permit acomparison of the sequence of said isolated nucleic acid molecule withthe sequence of DNA in said sample; and c) analyzing the result of saidcomparison.
 14. A plant, plant cell, plant material or seed of a plantwhich comprises a nucleic acid molecule according to claim 1 which isexogenous or heterologous to said plant or plant cell.
 15. A plant,plant cell, plant material or seed of a plant which comprises a vectorconstruct according to claim
 5. 16. A plant that has been regeneratedfrom a plant cell or seed according to claim
 14. 17. A plant, plantcell, plant material or seed of a plant which comprises a nucleic acidmolecule according to claim 1, wherein said plant has improved pHtolerance or phosphate use efficiency characteristics as compared to awild-type plant cultivated under the same conditions.
 18. A method forincreasing pH tolerance or phosphate use efficiency in a plantcomprising transforming a plant with a nucleic acid sequence accordingto claim
 1. 19. A transgenic plant having a gene construct comprising anucleic acid encoding a pH tolerance or phosphate use efficiencycomponent operably linked to a plant promoter so that the pH toleranceor phosphate use efficiency component is ectopically overexpressed inthe transgenic plant, and the transgenic plant exhibits: i) faster rateof growth, ii) greater fresh or dry weight at maturation, iii) greaterfruit or seed yield, iv) higher tolerance to pH, v) higher tolerance tolow phosphate concentration, or vi) higher tolerance to low nitrogenconcentration than a progenitor plant which does not contain thepolynucleotide construct, when the transgenic plant and the progenitorplant are cultivated under identical environmental conditions, whereinthe pH or phosphate use efficiency component is any one of thepolypeptides set forth in the Sequence Listing, or any one of theconsensus sequences in claim
 3. 20. A method for pH tolerance orphosphate use efficiency in a plant which comprises transforming a plantwith a nucleic acid sequence that encodes a polypeptide that comprisesat least one of the following: (a) an amino acid sequence that comprisesthe residues at positions 29-154 of the consensus sequence of Table 1-5,(b) an amino acid sequence that comprises the residues at positions18-128 of the consensus sequence of Table 2-6, (c) an amino acidsequence that comprises the residues at positions 57-230 of theconsensus sequence of Table 3-5, (d) an amino acid sequence thatcomprises the residues at positions 234-248 of the consensus sequence ofTable 3-5, and (e) an amino acid sequence that comprises the residues atpositions 10-276 of the consensus sequence of Table 4-1.
 21. A plant,plant cell, plant material of a plant with improved pH tolerance orphosphate use efficiency characteristics as compared to a wild-typeplant cultivated under the same conditions which comprises a nucleicacid sequence that encodes a polypeptide that comprises at least one ofthe following: (a) an amino acid sequence that comprises the residues atpositions 29-154 of the consensus sequence of Table 1-5, (b) an aminoacid sequence that comprises the residues at positions 18-128 of theconsensus sequence of Table 2-6, (c) an amino acid sequence thatcomprises the residues at positions 57-230 of the consensus sequence ofTable 3-5, (d) an amino acid sequence that comprises the residues atpositions 234-248 of the consensus sequence of Table 3-5, and (e) anamino acid sequence that comprises the residues at positions 10-276 ofthe consensus sequence of Table 4-1.